Vitamin D, via its active metabolite 1,25(OH)2D3, inhibits the proliferation and induces the differentiation of epidermal keratinocytes. 1,25(OH)2D3 binds to its receptor, the vitamin D receptor, (VDR) which mediates its biological effect by directly regulating target gene transcription. Two distinct coactivator complexes, DRIP (vitamin D receptor interacting proteins) and SRC (steroid receptor coactivator), are critical for VDR action. We have discovered that DRIP is the dominant coactivator complex in normal and transformed keratinocytes. However, following differentiation, SRC replaces DRIP as DRIP levels decline. Therefore, we postulate that normal keratinocyte differentiation requires the sequential transition from DRIP to SRC coactivation of VDR. Loss of this transition may result in resistance to the pro-differentiation effects of vitamin D in transformed keratinocytes. Therefore, we plan to determine the requirement for DRIP coactivation in comparison to SRC family coactivation during the differentiation process of keratinocytes. This will be achieved by over-expressing or inhibiting DRIP and SRC to selectively manipulate the function of these coactivators, and testing the effects on the ability of 1,25(OH)2D3 to regulate proliferation and differentiation. We will also determine the temporal sequence by which vitamin D regulated genes are specifically induced by the different coactivator complexes during the differentiation process, using chromatin immunoprecipitation. We will then determine the role of the LXXLL motifs of DRIP and SRC in mediating the ability of these coactivators to selectively regulate 1,25(OH)2D3 response, and aim to develop specific LXXLL derived peptides to manipulate the 1,25(OH)2D3 action on keratinocytes. Finally, the specificity of these manipulations on other nuclear receptors will be examined. These studies will clarify the molecular mechanism of vitamin D action on keratinocytes with the potential for finding therapeutic applications to treat hyper-proliferative dermatological diseases, such as psoriasis, and cutaneous malignancies.